Enhancement of production of NK cells from stem cells

ABSTRACT

A composition and a method for generating clinically safe NK cells derived from non-fully differentiated stem cells are provided. The non-fully differentiated stem cells are co-cultured with endogenous NK cells isolated from adipocyte-containing tissue to generate a high percentage of clinically safe NK cells, where anti-tumor activity of the clinically safe NK cells in vitro is similar to that of endogenous NK cells. Optimized Production of the clinically safe autologous NK cells from stem cells provides platform for treating cancer patients by applying an effective adoptive immunotherapy ranging from the early to terminal stages.

FIELD OF THE INVENTION

The field of the invention relates to compositions and methods forgenerating NK cells.

BACKGROUND

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

The American Cancer Society reports that more than 1.7 million newcancer cases will be diagnosed for 2019, and about 0.6 million Americanswill have died of cancer in 2019. The funding for cancer research inAmerica was more than 4 billion dollars in 2017 through the NationalCancer Institute, and researchers are making endless efforts to developeffective cancer treatments.

Immunotherapy is a very exciting approach to developing anti-tumortreatments. As a part of its normal function, the immune system detectsand destroys abnormal cancer cells, and immunotherapy helps the immunesystem to better act against cancer. One of these therapies is T-celltransfer therapy, which is a treatment that increases the naturalability of the patient's T cell to fight cancer. In this treatment, Tcells are collected from a patient and engineered, such that engineeredT cells are capable of detecting and binding to a specific antigen onthe surface of cancer cells through the expression of an antigen-bindingreceptor on the surface of T cells. The engineered T cells are givenback to the patient usually via a needle in the patient's vein(s). Thistherapy is called CAR (chimeric antigen receptor)-T cell therapy. CARTcells can detect and bind cancer cells effectively and efficiently. As aresult, CART cells can destroy cancer cells significantly more than theoriginal T cells, and the anti-tumor activity of CART cells has beenproven by many clinical trials.

Researchers have also experimented with generation of CARNK cells. Thereare some advantages of using NK cells rather than using T cells. First,unlike CART cells, CARNK cells retain an intrinsic capacity to detectand target cancer cells through their native receptors, so that it isless likely that cancer cells can escape from CAR cells bydownregulating the CAR target antigen expression. Second, it is lesslikely that patients will suffer from cytokine release syndrome becauseCARNK cells do not undergo clonal expansion within days to weeks. Thus,the level of cytokines released from CARNK is maintained within thenormal range. On the other hand, CART cells undergo clonal expansion,such that cytokine released from CART cells can reach a high level, andthis high level of cytokines may cause increased inflammation throughouta patient body. This can result in organ failure or even death.Consequently, researchers have started to utilize NK cells to develop aneffective anti-cancer treatment.

Because primary NK cells are difficult to isolate and purify,researchers have developed a protocol to generate NK cells using stemcells. These include induced pluripotent stem cells (iPSCs). Anadvantage of using iPSCs is that there is no immune rejection responsefrom the patient. The iPSCs derived NK cells can be released back to anindividual without concern for immune rejection because the iPSCs areobtained from the same individual. Additionally, there are alreadyestablished protocols to differentiate iPSCs to NK cells, for example,shown in U.S. Pat. No. 9,260,696.

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application werespecifically and individually indicated to be incorporated by reference.Where a definition or use of a term in an incorporated reference isinconsistent or contrary to the definition of that term provided herein,the definition of that term provided herein applies and the definitionof that term in the reference does not apply.

However, there is at least one significant disadvantage using iPSCs. Itis well known that undifferentiated iPSCs have intrinsically tumorigenicproperties. On the other hand, differentiated cells derived from iPSCslose tumorigenicity (“The tumourigenicity of iPSCs and theirdifferentiated derivates”, J. Cell. Mol. Med. Vol 17, No 6, P 782-791,2013). When iPSC derived NK cells contaminated with undifferentiatediPSCs are introduced to a patient, the undifferentiated iPSC maydifferentiate into cancer cells in a body of the patient. Therefore,iPSC derived NK cells must be completely separated from undifferentiatediPSCs. Because the NK cells must be completely separated fromundifferentiated iPSCs, a large amount of the NK cells are lost duringseparation of iPSCs from NK cells during the cell sorting process.

One way to reduce wasting large numbers of the generated NK cells is toincrease the rate of the differentiation into NK cells. U.S. Pat. No.9,260,696 teaches that, typically, only about half of stem cells (iPSCsand embryonic stem cells) are differentiated into NK cells in thepresence of inactivated antigen presenting cells.

Qu el al. have used adipose-derived stem cells (ADSCs) that aredifferentiated into NK cells “conversion of adipose-derived stem cellsinto Natural Killer-like cells with anti-tumor activities in nude mice,PLos ONE, Vol 4, e106246, 2014”. The efficiency of the ADSCdifferentiation to NK cells is not literally described, but in vitroresults indicate that the efficiency is at least 90%.

There are two known ways to generate ADSC-derived NK cells. One is touse sphere clusters and the other way is to express E4BP (NKcell-specific transcription factor) in ADSCs. In vitro anti-tumoractivity of NK cells developed from sphere clusters shows about 60% ofthe activity obtained from endogenous NK cells. On the other hand, invitro anti-tumor activity of NK cells expressing E4BP is similar to theactivity obtained from endogenous NK cells. This, in vitro anti-tumoractivity of the NK cells expressed with E4BP is greater than the NK cellderived from sphere clusters. However, E4BP3 is expressed in ADSCsthrough lentivirus transduction of E4BP3 to ADSCs. There is asignificant concern using such lentivirus exposed cells to treat cancerpatients.

Thus, there is still a need for efficiently generating clinically safeNK cells from stem cells, having at least similar in vitro anti-tumoractivity to the endogenous NK cells.

SUMMARY OF THE INVENTION

The inventive subject matter provides compositions and methods thatprovide a high percentage of stem cells that differentiate intoclinically safe NK cells, having at least similar (e.g. 60%, 70%, 80%,90%, 95%, or more than 95%) of the anti-tumor activity of endogenous NKcells in vitro.

The stem cells can include human induced pluripotent stem cells (iPSCs),adipocyte derived stem cells (ADSCs), embryonic stem cells, andhematopoietic stem cells.

The stem cells are used to create non-fully differentiated stem cells orundifferentiated NK precursor cells (These can be obtained by inducingthe stem cells to form CD34+ hematopoietic precursor cells)

In some embodiments, NK cells can be isolated from a cluster ofadipocytes. In other embodiments, NK cells are further purified from theStromal Vascular Fraction of ADSCs. Co-culturing non fullydifferentiated stem cells derived from stem cells with theadipocyte-isolated NK cells provides a high efficiency ofdifferentiation of stem cells into NK cells (e.g. greater than 50%, 60%,70%, 80%, 90%, or 95%). In some embodiments, stem cells can be derivedfrom the skin sample of individual and NK cells are derived from ADSCsof the same individual. In other embodiments, ADSCs can be isolated fromsuch a cluster of adipocytes,

In some embodiments, at least 60% of the non-fully differentiated stemcells are differentiated into clinically safe NK cells as a result ofco-culturing with the adipocyte NK cell. In preferred embodiments, atleast 75% of the non-fully differentiated stem cells are differentiatedinto clinically safe NK cells. In most preferred embodiments, at least90% of the non-fully differentiated stem cells are differentiated intoclinically safe NK cells as a result of co-culturing with the adipocyteNK cell.

In some embodiments, in vitro anti-tumor activity of stem cell derivedclinically safe NK cells is about 60% of the activity obtained fromendogenous NK cells. In preferred embodiments, the anti-tumor activityof stem cell derived clinically safe NK cells is about 70% of theactivity obtained from endogenous NK cells. In most preferredembodiments, the anti-tumor activity of stem cell derived clinicallysafe NK cells is about 90% of the activity obtained from endogenous NKcells.

In some embodiments, the co-culture does not include cytokines and/orgrowth factors.

The inventive subject matter also describes generating stem cell derivedclinically safe NK cells, including steps of 1) isolating NK cells froma cluster of adipocytes, 2) expanding stem cells, 3) inducing stem cellsto form non-fully differentiated stem cells 4) co-culturing thenon-fully differentiated stem cells with the NK cell, 5) inducingdifferentiation of the non-fully differentiated stem cells into NK cellsas a result of co-culturing. The co-culturing provides a high efficiencyof differentiation of stem cells into NK cells having similar anti-tumoractivity to endogenous NK cells. In some embodiments, a step ofisolating stem cell derived NK cells from a mixture of stem cell derivedNK cells and NK cells isolated from adipocytes is included.

The inventive subject matter further includes a method of treating acancer patient using clinically safe NK cells derived from stem cells,comprising the steps described in the method above, and furtherincluding 1) engineering a stem cell derived clinically safe NK cellinto a chimeric antigen receptor expressing NK (CARNK) cell, and 2)introducing the CARNK cell to a cancer patient.

As used herein, and unless the context dictates otherwise, the term“coupled to” is intended to include both direct coupling (in which twoelements that are coupled to each other contact each other) and indirectcoupling (in which at least one additional element is located betweenthe two elements). Therefore, the terms “coupled to” and “coupled with”are used synonymously.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided with respect to certain embodiments herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawing FIGURES in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depiction of a method according to the inventivesubject matter.

DETAILED DESCRIPTION

The following discussion provides many example embodiments of theinventive subject matter. Although each embodiment represents a singlecombination of inventive elements, the inventive subject matter isconsidered to include all possible combinations of the disclosedelements. Thus if one embodiment comprises elements A, B, and C, and asecond embodiment comprises elements B and D, then the inventive subjectmatter is also considered to include other remaining combinations of A,B, C, or D, even if not explicitly disclosed.

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

The recitation of ranges of values herein is merely intended to serve asa shorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein.

Unless the context dictates the contrary, all ranges set forth hereinshould be interpreted as being inclusive of their endpoints, andopen-ended ranges should be interpreted to include only commerciallypractical values. Similarly, all lists of values should be considered asinclusive of intermediate values unless the context indicates thecontrary.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

The current application provides compositions and methods in which ahigher percentage of stem cells are differentiated into NK cells.

Definitions

The term “stem cell” refers to special cells that have the ability todevelop into many different cell types, including muscle cells, braincells, and red and white blood cells. Stem cell-based therapies can beused to treat a variety of ailments including cancer, and many clinicaltrials based on stem cell therapies are on-going. Stem cells includeembryonic stem cells, adult stem cells including mesenchymal stem cells,hematopoietic stem cell, and adipocyte derived stem cells (ADSC), andinduced pluripotent stem cells (iPSCs) including ADSC-derived iPSCs, andectoderm/mesoderm/endoderm-derived stem cells.

The term “induced pluripotent stem cells” refers to a type ofpluripotent stem cells that can be generated directly from somaticcells. One advantage of using iPSCs is that there is no concern forimmune rejection. Indeed, iPSC developed from somatic cells obtainedfrom an individual can be applied to the same individual.

The term “lymphoid progenitor cells” or “undifferentiated NK cells”refers to CD34+ hematopoietic precursor cells that are derived from stemcells.

The term “non-fully differentiated stem (NFDS) cells” refers to stemcells and lymphoid progenitor cells.

The term “patient-specific adipocytes/NK cells” refers to theadipocyte/NK cells derived/isolated from the individual.

The term “clinically safe NK cells” refers to NK cells that do notprovide a clinically significant risk of introducing medical concernsthat are endogenously and/or intrinsically originated from the NK cells.Thus, exogenous effects of the NK cells, for example, immune rejectionand medical concerns as a result of environmental effects, are excluded.

The term “endogenous NK cells” refers to the NK cells that naturallydevelop into NK cells in the individual, including NK cells purified andisolated from the Stromal Vascular Fraction of ADSCs. NK cells in theblood stream are one example.

The term “in vivo” refers to a medical test, experiment, or procedurethat is done on/in a living organism, such as a laboratory animal orhuman.

The term “in vitro” refers to a medial study or experiment, which isdone in the laboratory within the confines of a test tube/laboratorydish.

Stem Cells

In some embodiments, adipocyte derived stem cells (ADSCs) can be used asstem cells (FIG. 1, 110 ). As described below, adipocyte residing NKcells can be isolated from human adipocyte-containing tissue obtainedfrom a patient's subcutaneous fat. In some embodiments the sameadipocyte-containing tissue can be used to isolate ADSCs. Taking fattissue from a patient and isolating ADSCs from the tissue is relativelystraightforward compared to isolating stem cells from bone marrow. Forexample, such tissue can be obtained by excision from a readilyaccessible subcutaneous site, or, alternatively, obtained by aliposuction procedure. It should be appreciated that a large number ofADSCs per volume of tissue, a high rate of proliferation, andanti-apoptotic function in ADSCs relative to other stem cells have beendemonstrated in various preclinical studies. Accordingly, the size of anadipose tissue sample for such a process can be relatively small (e.g.from about 1 cm³ to about 1,0000 cm³). In addition, as described above,it is already known that ADSCs are capable of differentiating into NKcells. Alternatively, suitable stem cells can be obtained from anypatient ectodermal, mesodermal, or endodermal tissue.

In some embodiments, iPSCs can be used as stem cells. As noted above,one advantage of the use of iPSCs is the minimal to no risk of immunerejection, as the iPSCs will be used to treat the individual that theyare obtained from. Such iPSCs can be generated from any suitableectodermal, mesodermal, or endodermal tissue. For example, fibroblastsisolated from a patient's skin can be used to generate suitable iPSCs.

It should be appreciated that the use of embryonic stem cells usuallycreates immune rejection because embryonic stem cells are not apatient-specific cells. Accordingly, use of iPSCs provides a significanttechnical advantage, especially for immunotherapy. The lack of rejectionpermits maintenance of immune function to be maintained to at least to anormal level.

In a preferred embodiment, iPSCs derived from ADSCs can be used. Amethod of generating such iPSC is taught in “Induced Pluripotent StemCells Generated from Human Adipose-Derived Stem Cells Using a Non-ViralPolycistronic Plasmid in Feeder-Free Conditions, PLoS ONE, 7(10), 2012”.

iPSCs Derived from Patient Specific Fibroblasts

In one example of a method of the inventive concept a virus-freepolycistronic plasmid method was applied to generate iPSCs from patientspecific fibroblasts s. Taking a virus free polycistronic plasmid,pIRES2-EGFP plasmid, as a basic backbone, four “Yamanaka” genes (humanOCT4, Sox2, Klf4, and c-Myc TF genes) were inserted into the vector inoskm order within a single open reading frame. The pIRES2-EGFP plasmidvector can be transfected into somatic cells without the need for viralpackaging. The iPSCs derived from ADSCs are detected in a feedercell-free environment with ectopic expression of the “Yamanaka” fourfactors. The iPSCs were further confirmed by the identification of majorES cell markers such as TRA-1-60, OCT4, Nanog and SSEA4 after 28 days ofculturing. The iPSCs formed teratomas that differentiated into all threegerm layers (i.e. endoderm, ectoderm, and mesoderm), indicating thatiPSCs derived from ADSCs were successfully generated.

iPSCs can be expanded, for example, using culture in laminin 521 coatedvessels, typically doubling in population within 7 days of culturing.Such expanded iPSCs can be banked for later use as described in “Lamininas a potent substrate for large-scale expansion of human inducedpluripotent stem cells in a closed cell expansion system, Stem cellsinternational, 2019”.

Some NK cells are found circulating in blood while others are tissueresidents. In the current subject matter, NK cells residing inadipocytes can be obtained by isolation of NK cells from apatient-specific adipocyte-containing tissue (“retained NK cellphenotype and functionality in non-alcoholic fatty liver disease,Frontiers in Immunology, Vol 10, 1255, 2019”). The tissue ismechanically dissociated (e.g. by enzymatic digestion in collagenase IIat 37° C.) and filtered through a 100 μm filter, and stained for thepresence of NK cell-specific markers (for example, CD56, CD3⁻, etc.).Cells with NK cell markers are separated from other cell types, forexample by flow cytometry.

Non-Fully Differentiated Stem Cells Co-Cultured with Adipocyte-IsolatedNK Cells

It should be appreciated that stem cells utilized in methods of theinventive concept can be totipotent or pluripotent (e.g. partiallydifferentiated but not committed to maturing into a single cell type).Within the context of this application the term “non-fullydifferentiated stem cells” is considered inclusive of totipotent,pluripotent, multipotent, and oligopotent stem cells. Non-fullydifferentiated stem cells including stem cells and lymphoid progenitorcells (FIG. 1, 110 a) can be cultured in DMEM-containing plastic culturedishes. The attached cells are allowed to reach 80% confluence. Theisolated NK cells from adipocyte-containing (FIG. 1, 120 ) can beapplied directly onto Non-fully differentiated stem cells in the culturedish, or they can be applied to a barrier (such as a divider or filter)that physically separates the stem cells from isolated NK cells (FIG. 1,130 ). In the presence of such a barrier the Non-fully differentiatedstem cells can, for example, be cultured on the surface of a bottom dishand NK cells isolated from adipocyte-containing tissue presented on atop culture dish. In such embodiments, molecules released from the NKcells can pass through the barrier and reach to the Non-fullydifferentiated stem cells. In some embodiments, cancer cells can beapplied on the top dish and/or surface of the barrier to activate NKcells isolated from adipocyte-containing tissue.

The ratio of lymphoid progenitor cell to a NK cell isolated fromadipocyte-containing tissue can range from 1:1 to 10,000:1. In someembodiments, the ratio is 1:1 to 1,000:1. In more preferred embodiments,the ratio is 1:1 to 1:10,000. Such co-culturing of these cells can bemaintained for from 8 hours to 2 months, typically at 37° C. and with 5%CO₂.

NK cells isolated from adipocyte-containing tissue can be healthy (withor without activation), or damaged NK cells. In some embodiments, asdescribed above, NK cells can be activated by co-culturing with cancercells in the presence of a barrier between non-fully differentiated stemcells and NK cells with cancer cells. In some embodiments, NK cells areinduced (activated) by introducing cytokines and/or growth factors (suchas IL-2, IL-3, IL-6, IL-7, IL-15, IL-18, IFN-γ, and/or TNF-α). In someembodiments, damaged NK cells, for example, having lower cytotoxicity,can be used for co-culturing with stem cells.

The duration of such co-culturing can range from overnight to 10 weeks.In preferred embodiments, the duration is 3 days to 8 weeks. In morepreferred embodiments, the duration is 1 week to 6 weeks. In the mostpreferred embodiments, the duration is 1 week to 4 weeks.

In some embodiments cell culture media used in such methods can includecytokines and/or growth factors. In other embodiments such cell culturemedia can exclude cytokines and/or growth factors. Suitable cytokinesand/or growth factors include IL-2, IL-3, IL-6, IL-7, IL-15, IL-18, stemcell factor, endothelial growth factor, granulocyte-macrophagecolony-stimulating factor, IFN-γ, and/or TNF-α.

Stem cells of the inventive concept can differentiate into various NKcells lines, including NK3.3, KHYG-1, NKL, NKYS, and NKT. The markersfor NK3.3 lines include CD2, CD11a, CD38, CD45, CD16, and CD56. Themarkers for KHYG-1 lines include CD2, CD3ε, CD7, CD8αα, CD33, CD56,CD122, and CD132. The markers for NKL lines include CD2, CD3ε, CD7,CD8αα, CD33, CD56, CD122, and CD132. The markers for NKT lines includeCD56, CD3. The markers for NKYS lines include CD2, CDS, CD7, and CD56.

Since all NK cell lines express CD56, and CD56 is specific to NK cells,in preferred embodiments cultured cells can be stained with CD 56 andseparated by cell sorting in a flow cytometer to separate NK cells(including both stem cell derived NK cells and NK cells isolated fromadipocyte-containing tissue) from undifferentiated stem cells.

Evaluation of Anti-Tumor Activity In Vitro

Cancer cells, for example, PC3 (human prostate cancer cell line, B CellLymphomas, HL-60 acute myeloid leukemia cell lines, U266 multiplemyeloma cell lines, U87 glioblastoma multiforme cell lines, A549non-small cell lung cancer cell lines, Saos-2 human osteosarcoma cancercell lines, A673 Ewing sarcoma cell line) and non-cancer cells (forexample, epithelial cells isolated from an individual) can befluorescently labeled using, for example, an activated fluorescent dyesuch as carboxyfluorescein diacetate succinimidyl ester (CFSE). Thecancer and/or non-cancer cells can be mixed with stem cell derived NKcells or NK cells isolated from adipocyte-containing tissue or blood atvarious ratios. The ratio of a cancer cells (and/or non-cancer cells) toNK cells can range from 1:1, 1:5, 1:10, 1:20: 1:50, and/or 1:100. Afterovernight incubation at 37° C. and 5% CO₂, cell culture can be stainedwith a dead cell marker (for example, propidium iodide or PI) and thenanalyzed by flow cytometry. The percentage of the dead cancer and/ornon-cancer cells are calculated by comparing the number of PI stainedcells to the number of CFSE stained cells.

Instead of using PI staining, cells can be observed microscopically todetermine viability. A phase-contrast and a fluorescent image can beobtained from the same location in such a cell culture and superimposed.Intact and lysed CF SE-labeled can be counted randomly for selectedimages. The percentage of the dead cancer or non-cancer cells arecalculated by comparing the number of stained lysed cells to the numberof stained lysed and stained intact cells.

Evaluation of Anti-Tumor Activity In Vivo

Tumor cells, for example, U87 glioblastoma multiforme cell lines, PC3(human prostate cancer), B Cell Lymphomas, HL-60 acute myeloid leukemiacell lines, U266 multiple myeloma cell lines, A549 non-small cell lungcancer cell lines, Saos-2 human osteosarcoma cancer cell lines, A673Ewing sarcoma cell line) can be subcutaneously injected into arepresentative number (e.g. about fifteen) 2-month-old male nude mice at1×10⁶ cells/mouse. One week later, the mice are separated into 3 groupsof approximately equal size. The first and second group areintravenously injected with 1×10⁷ cells of either stem cell derivedclinically safe NK cells or the endogenous NK cells specific to eachindividual mouse. The third group is a negative control. Tumor size canbe measured (e.g. with an electronic caliper, by image analysis, etc.)at regular intervals for up to 8 weeks after PC3 injection. The tumorsize can be calculated as length (mm)×width (mm²)×0.523.

Generated NK cells can be used for immunotherapy including CARNK therapy(FIG. 1, 140 ) against cancers and applied to a patient to treat theircancer (FIG. 1, 150 ). In addition, extracellular vesicles derived fromsuch NK cells can be used to produce an anti-tumor effect. The stem cellderived clinical safe NK cells can be used to obtain such extracellularvesicles for application to cancer therapy in humans. Therefore,generated NK cells described in the current subject matter are versatileenough to develop various treatments against cancers.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refer to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

What is claimed is:
 1. A cultured cell preparation, comprising: aplurality of non-fully differentiated stem cells; a first plurality ofNK cells that are endogenous NK cells obtained from adipocyte-containingtissue that comprises said endogenous NK cells; a barrier interposedbetween the plurality of non-fully differentiated stem cells and thefirst plurality of NK cells; and a second plurality of NK cells derivedfrom the plurality of non-fully differentiated stem cells.
 2. Thecultured cell preparation of claim 1, wherein the plurality of non-fullydifferentiated stem cells are selected from the groups consisting oflymphoid progenitor cells, human induced pluripotent stem cells (iPSCs),stem cells derived from the adipocyte-containing tissue (ADSCs),embryonic stem cells, and hematopoietic stem cells.
 3. The cultured cellpreparation of claim 2, wherein the iPSCs are iPSCs derived patientfibroblasts.
 4. The cultured cell preparation of claim 2, wherein theplurality of non-fully differentiated stem cells and the endogenous NKcells are derived from the same individual.
 5. The cultured cellpreparation of claim 1, wherein the first plurality of NK cells areisolated from a cluster of adipocytes.
 6. The cultured cell preparationof claim 5, wherein the first plurality of NK cells are isolated andpurified from Stromal Vascular Fraction of ADSCs.
 7. A method ofculturing NK cells, comprising: obtaining a first population of cellscomprising endogenous NK cells collected from a cluster of adipocytes;culturing non-fully differentiated stem cells; and, co-culturing thenon-fully differentiated stem cells with the plurality of NK cells witha barrier interposed between the non-fully differentiated stem cells andthe first population of NK cells to induce differentiation of thenon-fully differentiated stem cells into a second population of cellscomprising NK cells derived from the non-fully differentiated stemcells.
 8. The method of claim 7, wherein the non-fully differentiatedstem cells are selected from the group consisting of lymphoid progenitorstem cells, human induced pluripotent stem cells (iPSCs), stem cellsderived from adipocyte-containing tissue (ADSCs), embryonic stem cells,and hematopoietic stem cells.
 9. The method of claim 8, wherein theiPSCs are iPSCs derived from patient specific fibroblasts.
 10. Themethod of claim 8, wherein the non-fully differentiated stem cells andthe plurality of NK cells are derived from the same individual.
 11. Thecultured cell preparation of claim 1, wherein the plurality of non-fullydifferentiated stem cells is a plurality of induced pluripotent stemcell.
 12. The method of claim 7, wherein said non-fully differentiatedstem cells are induced pluripotent stem cells.
 13. The cultured cellpreparation of claim 1, further comprising a cancer cell.
 14. Thecultured cell preparation of claim 1, wherein the barrier is permeable.15. The method of claim 7, wherein the first population of cellscomprises a cancer cell.
 16. The method of claim 7, wherein the barrieris permeable.